Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C

Oscillations in cytosolic free Ca2+ concentration ([Ca2+](cyt)) are an important component of Ca2+-based signal transduction pathways. This fact has led us to investigate whether oscillations in [Ca2+](cyt) are involved in the response of stomatal guard cells to the plant hormone abscisic acid (ABA). We show that ABA induces oscillations in guard-cell [Ca2+](cyt). The pattern of the oscillations depended on the ABA concentration and correlated with the final stomatal aperture. We examined the mechanism by which ABA generates oscillations in guard-cell [Ca2+](cyt) by using 1-(6-{[17 beta-3-methoxyestra-1,3,5(10) -trien-17-yl] amino}-hexyl)-1H-pyrrole-2,5-dione (U-73122), an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC)-dependent processes in animals. U-73122 inhibited the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant PI-PLC, isolated from a guard-cell-enriched cDNA Library, in a dose-dependent manner. This result confirms that U-73122 is an inhibitor of plant PI-PLC activity. U-73122 inhibited both ABA-induced oscillations in [Ca2+](cyt) and stomatal closure. In contrast, U-73122 did not inhibit external Ca2+-induced oscillations in guard-cell [Ca2+](cyt) and stomatal closure. Furthermore, there was no effect of the inactive analogue 1-(6- {[17 beta-3-methoxyestra-1,3,5 (10)-trien-17-yl] amino} hexyl)-2,5-pyrrolidinedione on recombinant PI-PLC activity or ABA-induced and external Ca(2+-)induced oscillations in [Ca2+](cyt) and stomatal closure. This lack of effect suggests that the effects of U-73122 in guard cells are the result of inhibition of PI-PLC and not a consequence of nonspecific effects. Taken together, our data suggest a role for PI-PLC in the generation of ABA-induced oscillations in [Ca2+](cyt) and point toward the involvement of oscillations in [Ca2+](cyt) in the maintenance of stomatal aperture by ABA.